Exploring TCGA

Load dependencies

library(tidyverse)

── Attaching packages ──────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────── tidyverse 1.2.1 ──
✔ ggplot2 2.2.1     ✔ purrr   0.2.4
✔ tibble  1.4.2     ✔ dplyr   0.7.4
✔ tidyr   0.8.0     ✔ stringr 1.3.0
✔ readr   1.1.1     ✔ forcats 0.3.0
── Conflicts ─────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────── tidyverse_conflicts() ──
✖ dplyr::filter() masks stats::filter()
✖ dplyr::lag()    masks stats::lag()

library(cowplot)

Attaching package: ‘cowplot’

The following object is masked from ‘package:ggplot2’:

    ggsave

1 Reading & Cleaning

Define where the data lives

## Define data paths
in_path_varscan = 'data/maf/6c93f518-1956-4435-9806-37185266d248/TCGA.BRCA.varscan.6c93f518-1956-4435-9806-37185266d248.DR-10.0.somatic.maf.gz'
in_path_muse = 'data/maf/b8ca5856-9819-459c-87c5-94e91aca4032/TCGA.BRCA.muse.b8ca5856-9819-459c-87c5-94e91aca4032.DR-10.0.somatic.maf.gz'
in_path_ss = 'data/maf/7dd592e3-5950-4438-96d5-3c718aca3f13/TCGA.BRCA.somaticsniper.7dd592e3-5950-4438-96d5-3c718aca3f13.DR-10.0.somatic.maf.gz'
in_path_mutect = 'data/maf/995c0111-d90b-4140-bee7-3845436c3b42/TCGA.BRCA.mutect.995c0111-d90b-4140-bee7-3845436c3b42.DR-10.0.somatic.maf.gz'

Reading data

df = read_tsv(
  in_path_mutect,
  comment = '#'
)

Parsed with column specification:
cols(
  .default = col_character(),
  Entrez_Gene_Id = col_integer(),
  Start_Position = col_integer(),
  End_Position = col_integer(),
  t_depth = col_integer(),
  t_ref_count = col_integer(),
  t_alt_count = col_integer(),
  n_depth = col_integer(),
  ALLELE_NUM = col_integer(),
  DISTANCE = col_integer(),
  TRANSCRIPT_STRAND = col_integer(),
  GMAF = col_double(),
  AFR_MAF = col_double(),
  AMR_MAF = col_double(),
  EAS_MAF = col_double(),
  EUR_MAF = col_double(),
  SAS_MAF = col_double(),
  AA_MAF = col_double(),
  EA_MAF = col_double(),
  PUBMED = col_integer(),
  PICK = col_integer()
  # ... with 13 more columns
)
See spec(...) for full column specifications.

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number of columns of result is not a multiple of vector length (arg 1)203 parsing failures.
row # A tibble: 5 x 5 col     row col   expected               actual file                                                                                                                         expected   <int> <chr> <chr>                  <chr>  <chr>                                                                                                                        actual 1  1941 PHENO no trailing characters ;0     'data/maf/995c0111-d90b-4140-bee7-3845436c3b42/TCGA.BRCA.mutect.995c0111-d90b-4140-bee7-3845436c3b42.DR-10.0.somatic.maf.gz' file 2  3843 PHENO no trailing characters ;0     'data/maf/995c0111-d90b-4140-bee7-3845436c3b42/TCGA.BRCA.mutect.995c0111-d90b-4140-bee7-3845436c3b42.DR-10.0.somatic.maf.gz' row 3  4468 PHENO no trailing characters ;0     'data/maf/995c0111-d90b-4140-bee7-3845436c3b42/TCGA.BRCA.mutect.995c0111-d90b-4140-bee7-3845436c3b42.DR-10.0.somatic.maf.gz' col 4  6145 PHENO no trailing characters ;0     'data/maf/995c0111-d90b-4140-bee7-3845436c3b42/TCGA.BRCA.mutect.995c0111-d90b-4140-bee7-3845436c3b42.DR-10.0.somatic.maf.gz' expected 5  9783 PHENO no trailing characters ;1     'data/maf/995c0111-d90b-4140-bee7-3845436c3b42/TCGA.BRCA.mutect.995c0111-d90b-4140-bee7-3845436c3b42.DR-10.0.somatic.maf.gz'
... ................................. ... ........................................................................................................................................................................ ........ ..................................................................................................................................................................................................................................................................................................... ...... ................................................................................................................................................................................................. .... ................................................................................................................................................................................................. ... ................................................................................................................................................................................................. ... ................................................................................................................................................................................................. ........ .................................................................................................................................................................................................
See problems(...) for more details.

Preview data

print(dim(df))

[1] 120988    120

Warning messages:
1: Unknown or uninitialised column: 'GENE'. 
2: Unknown or uninitialised column: 'GENE'. 
3: Unknown or uninitialised column: 'GENE'. 
4: Unknown or uninitialised column: 'GENE'. 
5: Unknown or uninitialised column: 'GENE'. 

head(df, 10)

Read & concatenate multiple files

## Define column types
col_types = cols_only(
  Chromosome = 'c',
  Start_Position = 'i',
  End_Position = 'i',
  SYMBOL = 'c',
  Reference_Allele = 'c',
  Allele = 'c', 
  Variant_Classification = 'c', 
  IMPACT = 'c',
  Variant_Type = 'c',
  Tumor_Sample_Barcode = 'c'
)

Warning message:
Unknown or uninitialised column: 'GENE'. 

## Concatenate vertically
in_paths = c(
  in_path_varscan,
  in_path_muse,
  in_path_ss,
  in_path_mutect
)
df = bind_rows(lapply(
  in_paths, 
  read_tsv, 
  col_types = col_types,
  comment = '#'
))

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## Convert characters to factors
# Note, if we weren't concatenating,
# this could be done while reading 
# by specifying 
# `COL_NAME = col_factor(levels=NULL)`
# inside cols_only()
df = mutate_if(
  df,
  is.character,
  as.factor
)
print(dim(df))

[1] 370461     10

head(df)

Rename and reorder columns

## Rename
col_name_map = list(
  CHR = 'Chromosome',
  START = 'Start_Position',
  END = 'End_Position',
  GENE = 'SYMBOL',
  REF = 'Reference_Allele',
  ALT = 'Allele',
  CLASS = 'Variant_Classification',
  IMPACT = 'IMPACT',
  TYPE = 'Variant_Type',
  BARCODE = 'Tumor_Sample_Barcode'
)
df = rename(df, !!! col_name_map)
## Reorder
keep_cols = names(col_name_map)
df = select(df, keep_cols)
head(df)

Fill missing data

levels(df$GENE) = c(levels(df$GENE), 'INTERGENIC')
df$GENE[is.na(df$GENE)] = 'INTERGENIC'

Create new columns from string ops

df$SAMPLE = str_sub(df$BARCODE, 1, 12)
df$MUTATION = paste(
  df$CHR, 
  df$GENE,
  df$START,
  df$END,
  df$REF,
  df$ALT,
  sep = ':'
)
## Convert new columns to factors
## if applicable
df$SAMPLE = as.factor(df$SAMPLE)
df$MUTATION = as.factor(df$MUTATION)

Remove duplicates

## Remove duplicate rows
df = unique(df)
## Remove duplicate based on column
df_mut = df[!duplicated(df$MUTATION),]
print(dim(df))

[1] 132916     12

print(dim(df_mut))

[1] 130625     12

Remove columns

drop_cols = c('BARCODE', 'SAMPLE')
df_mut = select(df_mut, -one_of(drop_cols))
print(dim(df_mut))

[1] 130625     10

head(df_mut)

Writing files

write_delim(
  df, 
  'data/out/df_r.tsv', 
  delim = '\t'
)

Read & write binary files

rds_path = 'data/out/df.rds'
## Write
saveRDS(df, rds_path)
## Read
df = readRDS(rds_path)

Long to wide

rr

Shorten to relevant columns

Note, we could include other columns here

like GENE and IMPACT, but we won’t

to match the python example.

df_tmp = select(df, c(‘SAMPLE’, ‘MUTATION’)) df_tmp$EXISTS = 1

df_wide = spread( df_tmp, key = ‘SAMPLE’, value = ‘EXISTS’, fill = 0 )

rm(df_tmp)

print(dim(df_wide)) head(df_wide)

Add info from 1 df to another (merge)

rr

Select columns to add on and col to join on

cols = c(‘MUTATION’, ‘GENE’, ‘IMPACT’)

Merge

df_wide = merge( df_wide, select(df_mut, cols), by = ‘MUTATION’, all.x = TRUE )

Re-order columns

Put new columns first

df_wide = select(df_wide, cols, everything())

Note, first 3 columns are out of order

w/ python example.

print(dim(df_wide)) head(df_wide)

Wide to long

rr

Set value and id columns

value_cols = grep(‘TCGA-’, names(df_wide), value = TRUE)

Melt

df_long = gather( df_wide, key = ‘SAMPLE’, value = ‘EXISTS’, value_cols )

Clean up

df_long = filter(df_long, EXISTS != 0) df_long = select(df_long, -EXISTS)

print(dim(df_long)) head(df_long)

Free memory

rr rm(df_wide, df_long)

Counting

Count unique elements

n_genes = length(unique(df$GENE))
n_samples = length(unique(df$SAMPLE))
n_mutations = length(unique(df$MUTATION))
n_mutation_classes = length(unique(df$CLASS))
print('Number of unique')

[1] "Number of unique"

cat('\tSamples: ', n_samples, '\n')

    Samples:  986 

cat('\tGenes: ', n_genes, '\n')

    Genes:  19168 

cat('\tMutations: ', n_mutations, '\n')

    Mutations:  130625 

cat('\tMutation Classes: ', n_mutation_classes, '\n')

    Mutation Classes:  18 

Count factor levels

class_counts = sort(summary(df$CLASS), decreasing = TRUE)
impact_counts = sort(summary(df$IMPACT), decreasing = TRUE)
type_counts = sort(summary(df$TYPE), decreasing = TRUE)
print('Counts per CLASS:')

[1] "Counts per CLASS:"

print(class_counts)

     Missense_Mutation                 Silent                  3'UTR                 Intron 
                 66371                  23881                  11052                   6990 
     Nonsense_Mutation        Frame_Shift_Del                  5'UTR                    RNA 
                  6056                   3661                   3392                   2543 
       Frame_Shift_Ins            Splice_Site          Splice_Region                3'Flank 
                  1975                   1921                   1537                   1132 
               5'Flank           In_Frame_Del           In_Frame_Ins       Nonstop_Mutation 
                   906                    869                    433                     93 
Translation_Start_Site                    IGR 
                    90                     14 

print('Counts per IMPACT:')

[1] "Counts per IMPACT:"

print(impact_counts)

MODERATE MODIFIER      LOW     HIGH 
   67648    26059    25413    13796 

print('Counts per TYPE:')

[1] "Counts per TYPE:"

print(type_counts)

   SNP    DEL    INS 
121319   7316   4281 

## Display some instances
# Need to override the maxsum = 50 arg
mut_counts =  sort(
  summary(df$MUTATION, maxsum = n_mutations), 
  decreasing = TRUE)
print('Top repeated (>10) mutations:')

[1] "Top repeated (>10) mutations:"

print(mut_counts[mut_counts > 10])

     chr3:PIK3CA:179234297:179234297:A:G      chr3:PIK3CA:179218303:179218303:G:A 
                                     121                                       63 
     chr3:PIK3CA:179218294:179218294:G:A chr1:ST6GALNAC3:76576946:76576947:-:AAAC 
                                      43                                       33 
      chr14:AKT1:104780214:104780214:C:T         chr10:GATA3:8069470:8069471:CA:- 
                                      25                                       21 
       chr3:MUC4:195783009:195783009:C:T           chr17:TP53:7675088:7675088:C:T 
                                      21                                       20 
       chr3:MUC4:195783008:195783008:A:G     chr15:GOLGA6L6:20535018:20535018:C:T 
                                      20                                       17 
     chr3:PIK3CA:179203765:179203765:T:A      chr3:PIK3CA:179234297:179234297:A:T 
                                      17                                       13 
 chr6:OPRM1:154107953:154107958:TTTTTA:-           chr17:TP53:7673802:7673802:C:T 
                                      13                                       12 
      chr16:PKD1P6:15104542:15104542:T:A      chr1:NBPF12:146963189:146963189:G:C 
                                      11                                       11 

sample_counts =  sort(
  summary(df$SAMPLE, maxsum = n_samples), 
  decreasing = TRUE)
print('Samples with most mutations:')

[1] "Samples with most mutations:"

print(sample_counts[0:10])

TCGA-AN-A046 TCGA-AC-A23H TCGA-5L-AAT1 TCGA-BH-A18G TCGA-AN-A0AK TCGA-A8-A09Z TCGA-BH-A0HF 
        7948         6711         2117         2033         2029         1924         1695 
TCGA-AO-A128 TCGA-D8-A1XK TCGA-BH-A0B6 
        1644         1541         1419 

print('Samples with least mutations:')

[1] "Samples with least mutations:"

print(tail(sample_counts, 10))

TCGA-A2-A0ES TCGA-AC-A2FB TCGA-AR-A24W TCGA-AR-A252 TCGA-LL-A440 TCGA-AO-A1KO TCGA-A2-A1G6 
          18           17           16           16           16           15           12 
TCGA-A8-A08C TCGA-A2-A25F TCGA-AC-A2FK 
           9            7            2 

Summarize by group

df_summary = df %>%
  group_by(GENE) %>%
  summarise(
    N_MUTATIONS = n(),
    N_UNIQUE_MUTATIONS = n_distinct(MUTATION),
    N_SAMPLES = n_distinct(BARCODE)
)
head(df_summary)

Sorting

df_summary = df_summary %>%
  arrange(desc(N_SAMPLES), N_MUTATIONS)
head(df_summary)

Summarize by multiple groups

df_impact = df %>%
  group_by(GENE, IMPACT) %>%
  summarise(COUNT = n_distinct(MUTATION))
print(dim(df_impact))

[1] 46692     3

head(df_impact)

Visualize

Use cowplot for nice theme and subplotting https://cran.r-project.org/web/packages/cowplot/vignettes/introduction.html

img_dir = 'data/out/img/'

Ranking

NOTE: shape = ‘4’ corresponds to X’s

x = 0:(n_samples-1)
y = unname(sample_counts)
p = ggplot(mapping = aes(x, y)) +
  geom_line() +
  geom_point(shape = 4) +
  ggtitle('Mutations per Individual') +
  xlab('Individual Rank') +
  ylab('Number of Mutations')
 
p
ggsave(paste0(img_dir, 'rank_r.png')) 

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Bar plot

p = ggplot(
    mapping = aes(names(class_counts), unname(class_counts))
  ) +
  geom_bar(stat = 'identity') +
  scale_x_discrete(limits = rev(names(class_counts))) +
  coord_flip()
p
ggsave(paste0(img_dir, 'bar_r.png'))

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Subplots

## First plot
# Reuse instance from above
p1 = p
## Second plot
n_genes_to_plot = 5
genes = df_summary$GENE[0:n_genes_to_plot]
counts = df_summary$N_SAMPLES[0:n_genes_to_plot]
p2 = ggplot(
    mapping = aes(genes, counts)
  ) +
  geom_bar(stat = 'identity') +
  scale_x_discrete(limits = genes)
  
p_grid = plot_grid(p1, p2, ncol = 2)
save_plot(paste0(img_dir, 'subplot_r.png'), p_grid, ncol = 2)

Count vs identity

df_identity = df_summary %>%
  arrange(-N_MUTATIONS)
df_count = df
# Plot params
n_genes_to_plot = 5
genes = df_identity$GENE[0:n_genes_to_plot]
counts = df_identity$N_MUTATIONS[0:n_genes_to_plot]
# Identity plot
p1 = ggplot(mapping = aes(genes, counts)) +
  geom_bar(stat = 'identity') + 
  scale_x_discrete(limits = genes) +
  ggtitle('# Mutations - Identity')
# Count plot
p2 = ggplot(df, aes(GENE)) +
  geom_bar() +
  scale_x_discrete(limits = genes) + 
  ggtitle('# Mutations - Count')
p_grid = plot_grid(p1, p2, ncol = 2)

Removed 131195 rows containing non-finite values (stat_count).

save_plot(paste0(img_dir, 'count_vs_identity_r.png'), p_grid, ncol = 2)
p_grid

Plot with grouping

## Plot params
n_genes_to_plot = 5
genes = df_summary$GENE[0:n_genes_to_plot]
## Filter to relvant data
df_plt = df_impact %>%
  filter(GENE %in% genes)
## Reorder for plot
# This is an alternative to 
# scale_x_discrete(limits=genes)
df_plt$GENE = factor(df_plt$GENE, levels = genes)
p = ggplot(data = df_plt) +
  geom_bar(
    aes(x=GENE, y=COUNT, fill=IMPACT),
    stat = 'identity',
    position = 'dodge'
  ) +
  scale_fill_brewer(
    type='qual',
    palette = 'Set1'
  ) +
  ggtitle('Impact of Mutations')
p
ggsave(paste0(img_dir, 'group_r.png'), p)

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Faceted plot

## Get some samples and genes
samples = names(sample_counts[0:2])
genes = c('PIK3CA', 'TTN', 'MUC16')
## Filter to relevant data
df_plt = df %>%
  filter(SAMPLE %in% samples & GENE %in% genes)
## Reorder
df_plt$GENE = factor(df_plt$GENE, levels = genes)
df_plt$SAMPLE = factor(df_plt$SAMPLE, levels = samples)
## Plot
p = ggplot(df_plt) +
  geom_bar(
    aes(x=GENE, fill=IMPACT),
    position = 'dodge'
  ) + 
  scale_fill_brewer(
    type='qual',
    palette = 'Set1'
  ) +
  facet_wrap('SAMPLE')
p
ggsave(paste0(img_dir, 'facet_r.png'), p)

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